The long range goal of this proposal is to gain insight into the role of the cell membrane skeleton in human diseases. Defects in the erythrocyte membrane skeleton integral proteins underlie some hereditary hemolytic anemias in humans and mice, and recent studies implicate neuronal membrane skeletal proteins in neurodegenerative processes in aging and in Alzheimer's disease. However, little is known about the structure, function and regulatory processes of the neuronal cell membrane skeleton and its major component - brain spectrin. Neuronal compartmentalization of brain spectrin isoforms into axons and presynaptic terminals (nonerythroid spectrin) and into cell body and dendrites (erythroid spectrin) suggests that brain spectrin isoforms may perform related but distinct functions in neuronal cells. The proposed work on spectrin tetramer formation and the spectrin SH3 domain should give us the basis to understand functional differences between brain spectrin isoforms. The spectrin tetramer is the basic functional unit of the membrane skeleton and its disruption affects the spectrin-actin interaction in vitro. We propose to analyze the tetramer formation of different brain spectrin isoforms using recombinant alpha- and beta-spectrin polypeptides that contain the regions of spectrin most likely to be involved in tetramer formation. The interaction will be studied using established in vitro functional binding assays and site-directed mutagenesis to obtain deletions and/or single amino acid substitutions in the functional recombinant polypeptides. The specific regions of spectrin subunits involved in the interaction will be determined. We will also address the question whether different isoforms of brain spectrin are able to form heteromeric complexes in vitro. It has been proposed that the SH3 domain plays a role in signal transduction and regulation of the membrane skeleton assembly. Recent identification of several proteins binding to the SH3 domain of tyrosine kinases, Grb2 and PLC-gamma suggests that the spectrin SH3 domain may also function through a binding to a known protein(s) since the SH3 ligand- binding site is conserved among several already known proteins. Alternatively, the brain spectrin SH3 domain may bind a novel protein. To address the function of the brain spectrin SH3 domain we will identify and clone a protein(s) which binds to the erythroid and nonerythroid alpha- spectrin SH3 domain using established recombinant DNA techniques. Protein ligands interacting with the spectrin SH3 domain are likely to form complexes with spectrin in vivo in cells. To identify these potential physiological interactions of brain spectrin SH3 domain we will use GST- affinity agarose and immunoprecipitation to identify SH3-binding proteins in cell lysates from neuronal cell lines naturally expressing spectrin and from transfected cell lines.